Posted by
Johannes Helm on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Reporting-laser-power-in-publication-tp7584120p7584123.html
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Dear Jolien,
it is not an easy task to measure the power in the focal region of the
objective "exactly" and the job gets even more complicated if you want
to determine the power "per pixel" in the way you describe.
1)
In general:
a)
Unless you have a dry objective, some power will, even - and
particularly - in case of "perfect" or "quasi perfect" alignment - be
"lost" when measuring the laser power with the detector placed under the
objective because of total reflection (unless you could "immerse" the
sensitive area of the power meter in the immersion medium, which most
probably would be bad for the power meter). In other words: If you have
an oil immersion objective and do not have any oil on its front lens,
the "outer ring" of light incident from the inside of the objective to
the front lens will be lost due to total reflection (will be "sent back
into the objective").
b)
If you have two objectives of the same type - and preferrably of the
same batch - it is possible to measure the power losses in two
objectives on an optical bench. Divide the rate of loss by two and you
will have the transmission losses in one objective. During this
measurement, you will have to make sure that the optical axes of the two
objectives are (quasi)collinear, that the distance between to two outer
front lens surfaces matches twice the focal distance of the objective
type and that there is immersion medium between the two front lenses.
The latter can be a practical problem when you have lenses with large
working distances; in this case a "vertical optical" bench would be more
suitable (or a workshop made objectve holder to be placed in the
position, where the microscope condenser usually is, so that the second
objective would establish the condenser under which you could place the
light detector).
c)
Find out about the diameter of the image size aperture of the
objective. Make sure the laser beam is collinear to the optical axis of
the microscope (by centering the scanning mirrors) and is expanded at
least so much that it overfills this diameter and have a ring made in
your workshop to be screwed into the objective revolver and with an
aperture as large as the image size aperture of the objective. Measure
the laser power under this ring. This value tells you, how much laser
power is incident onto the objective "from the rear side". Multiply this
value with the transmission rate of the objective and then you know how
much power exits from the objective and will be incident onto the
specimen. (At all these power measurements, you will have to select the
correct wavelength for the power meter.)
2)
In particular:
a)
You canNOT, if you want to do a "really correct job", simply take the
power and divide by the pixel size. Most objectives will have an
un-negligible curvature of field. If your specimen is "perfectly flat",
not all the spots on the flat surface of the specimen will receive the
fully focussed laser beam at the same time. In other words: If you focus
onto a landmark structure in the center of the field of view, the focus
close to the periphery of the field of view will be at a slightly
different axial position. In many cases this would not be a problem.
However, you should not assume that the curvature of the field of view
is zero across the entire field of view.
b)
Many - although not all - scanning systems exhibit vignetting effects.
The farer you approach "the corners of the image", the less power will
be transmitted to the specimen since part of the beam will be "lost"
because of beam vignetting. There are ways to estimate these losses
pretty well (e.g. scanning uniformly reflective specimens AT WHICH YOU
MUST BE VERY CAREFUL TO NOT DESTROY THE DETECTORS) and comparing signal
strengths at the different pixel positions.
3)
I would ask "my user" which tolerances in the power measurement she or
he would be willing to accept. Quite naturally, the narrower the
measurement uncertainty intervals are, which she or he would be willing
to accept, the more complicated the job will get. If she or he needs
something like "100 microwatts plus minus 30 microwatts", then forget
all my comments mentioned above. If delta(P) should be in the percent
region, then the job is, indeed, quite complicated. It is to my personal
mind, anyway, "not quite comme il faut" to write in a publication in a
scientific paper a "number" without appropriate error margins.
Nevertheless, this is accepted by many journals as far as "technical"
numbers in the "Materials and Methods" paragraph are concerned.
Best wishes,
Johannes
On 2015-08-14 19:08, Jolien Tyler wrote:
> *****
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>
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> posting.
> *****
>
> Hello All,
>
> I have a user who is examining heat shock proteins and, long story
> really
> short, is using our laser scanning confocal to blast ROIs of with 405
> nm
> laser light and examining hsp response. I'm trying to determine how
> best
> to report the amount of "blasting" being done to the samples.
>
> I have a power meter that I can place at the sample plane to collect
> power
> in milliwatts accounting for the optical light path/objective. I know
> the
> pixel size in microns being imaged. The power meter result is per
> pixel
> since this is a laser scanning confocal, regardless of the size of the
> power meter detector (correct?). Thus, I should be able to simply
> divide
> the power meter result and by pixel area to get mW per square micron
> (irradiance). Or am I totally off-track here?
>
> Thanks,
> Jolien
>
>
>
> ---------------------------------------------
> Jolien Tyler, Ph.D.
>
> University of Colorado
> MCD Biology
> 347 UCB
> Boulder, CO 80309
> 303-492-5955 (office)
>
>
http://mcdb.colorado.edu/facilities/lmcf/index.html--
P. Johannes Helm
Voice: (+47) 228 51159 (office)
Fax: (+47) 228 51499 (office)